Claims for Patent: 8,603,761

Summary for Patent: 8,603,761

Title:	GLP-1 receptor agonist bioassay
Abstract:	Provided herein are cell-based bioassays for measuring GLP-I receptor agonist activity of test compounds, such as GLP-I receptor agonist compounds. Exemplary GLP-I receptor agonist compounds include exendins, exendin analogs, GLP-1(7-37), and GLP-1(7-37) analogs. The bioassays are useful for quantitatively determining cAMP generated in samples containing GLP-I receptor agonist compounds (e.g., exenatide) and 6-23 (clone 6) cells having GLP-I receptors, whereby the amount of cAMP generated can be correlated to the GLP-I receptor agonist activity of the GLPI receptor agonist compounds (e.g., exenatide). Suitable cell-based bioassays include enzyme-linked immunosorbent assays and homogeneous time-resolved fluorescence assays.
Inventor(s):	Nicolaou; Michalis (San Diego, CA), Bancroft; Frederick Charles (Cardiff, CA), Herich; John Patrick (Cardiff, CA), Lwin; Aung Naing (Carlsbad, CA), Gupta; Swati (San Diego, CA)
Assignee:	Amylin Pharmaceuticals, LLC (San Diego, CA) AstraZeneca Pharmaceuticals LP (Wilmington, DE)
Application Number:	13/691,421
Patent Claims:	1. A method for detecting or measuring glucagon-like peptide-1 (GLP-1) receptor agonist activity of a test compound comprising: (a) preparing a sample comprising (i) a test compound; (ii) 6-23 (clone 6) cells; and (iii) a detectably labeled cAMP; (b) forming a reaction mixture by combining the sample from step (a) and an anti cAMP antibody; (c) quantitatively measuring cAMP in the sample; and (d) correlating the amount of cAMP in the sample to the GLP-1 receptor agonist activity of the test compound. 2. The method of claim 1, wherein the sample comprises: (i) a pharmaceutical composition which comprises the test compound; (ii) 6-23 (clone 6) cells; and (iii) a detectably labeled cAMP. 3. The method of claim 1, further comprising incubating the reaction mixture. 4. The method of claim 1, wherein the test compound is a GLP-1 receptor agonist compound. 5. The method of claim 1, wherein the test compound is exenatide. 6. The method of claim 1, wherein the test compound is liraglutide; albiglutide; taspoglutide; LY2189265; LY2428757; lixisenatide; CJC-1134; [N.sup..epsilon.-(17-carboxyheptadecanoic acid)Lys.sup.20]exendin-4-NH.sub.2; [N.sup..epsilon.-(17-carboxy-heptadecanoyl)Lys.sup.32]exendin-4-NH.sub.2; [desamino-His.sup.1,N.sup..epsilon.-(17-carboxy-heptadecanoyl)Lys.sup.20]- exendin-4-NH.sub.2; [Arg.sup.12,27,NLe.sup.14, N.sup..epsilon.-(17-carboxyhepta-decanoyl)Lys.sup.32]exendin-4-NH.sub.2; [N.sup..epsilon.-(19-carboxynonadecanoylamino)Lys.sup.20]-exendin-4-NH.su- b.2; [N.sup..epsilon.-(15-carboxypentadecanoylamino)Lys.sup.20]-exendin-4-- NH.sub.2; [N.sup..epsilon.-(13-carboxytridecanoylammo)Lys.sup.20]exendin-4- -NH.sub.2; [N.sup..epsilon.-(11-carboxy-undecanoylamino)Lys.sup.20]exendin- -4-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-AEEA-MPA)-NH.sub.2; exendin-4-Lys.sup.40(s-AEEA-AEEA-MPA)-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-AEEA-MPA)-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-MPA)-albumin; exendin-4-Lys.sup.40(.epsilon.-AEEA-AEEA-MPA)-albumin; exendin-4-Lys.sup.40(.epsilon.-AEEA-MPA)-albumin; GLP-1(7-38); desamino-His.sup.7,Arg.sup.26, Lys.sup.34(.gamma.-Glu(N-.alpha.-hexadecanoyl)))-GLP-1(7-37); desamino-His.sup.7,Arg.sup.26,Lys.sup.34,(N.sup..epsilon.octanoyl)-GLP-1(- 7-37); Arg.sup.26,34,Lys.sup.38(N.sup..epsilon.-(.omega.carboxypentadecano- yl))-GLP-1(7-38); Arg.sup.26,34,Lys.sup.36(N.sup..epsilon.-(.gamma.-Glu(N-.alpha.-hexadecan- oyl)))-GLP-1(7-37); or any peptide comprising the amino acid sequence of any one of SEQ ID NOs:1-40 and 43-45; wherein the peptide comprising the amino acid sequence of any one of SEQ ID NOs:25-40 may optionally be linked to the amino acid sequence of SEQ ID NO:42; wherein MPA and AEEA refer to maleimidopropionic acid and [2-(2-amino)ethoxy)lethoxy acetic acid, respectively; and wherein the test compound is optionally amidated. 7. A method for detecting or measuring GLP-1 receptor agonist activity of a test compound comprising: (a) preparing a sample comprising a test compound and 6-23 (clone 6) cells; (b) forming a reaction mixture by combining (i) the sample from step (a); (ii) an anti-cAMP antibody linked to a rare earth cryptate or a rare earth chelate; and (iii) cAMP linked to a fluorescent moiety; (c) irradiating the reaction mixture from step (b); (d) quantitatively determining the amount of cAMP in the sample; and (e) correlating the amount of cAMP in the sample to the GLP-1 receptor agonist activity of the test compound. 8. The method of claim 7, wherein the sample comprises (i) a pharmaceutical composition which comprises the test compound and (ii) 6-23 (clone 6) cells. 9. The method of claim 7, further comprising incubating the reaction mixture. 10. The method of claim 7, wherein the anti-cAMP antibody is linked to a terbium cryptate, a europium cryptate, a dysprosium cryptate, a samarium cryptate, a neodymium cryptate, a terbium chelate, a europium chelate, a dysprosium chelate, a samarium chelate, a neodymium chelate, or a combination of two or more thereof. 11. The method of claim 7, wherein the anti-cAMP antibody is an anti-cAMP monoclonal antibody. 12. The method of claim 7, wherein the fluorescent moiety is allophycocyanin, allophycocyanin B, phycocyanin C, phycocyanin R, or a combination of two or more thereof. 13. The method of claim 7, wherein the fluorescent moiety is a crosslinked allophycocyanin. 14. The method of claim 7, wherein the test compound is a GLP-1 receptor agonist compound. 15. The method of claim 7, wherein the test compound is exenatide. 16. The method of claim 7, wherein the test compound is liraglutide; albiglutide; taspoglutide; LY2189265; LY2428757; lixisenatide; CJC-1134; [N.sup..epsilon.-(17-carboxyheptadecanoic acid)Lys.sup.20]exendin-4-NH.sub.2; [N.sup..epsilon.-(17-carboxy-heptadecanoyl)Lys.sup.32]exendin-4-NH.sub.2; [desamino-His.sup.1,N.sup..epsilon.-(17-carboxy-heptadecanoyl)Lys.sup.20]- exendin-4-NH.sub.2; [Arg.sup.12,27,NLe.sup.14,N.sup..epsilon.-(17-carboxyhepta-decanoyl)Lys.s- up.32]exendin-4-NH.sub.2; [N.sup..epsilon.-(19-carboxynonadecanoylamino)Lys.sup.20]-exendin-4-NH.su- b.2; [N.sup..epsilon.-(15-carboxypentadecanoylamino)Lys.sup.20]-exendin-4-- NH.sub.2; [N.sup..epsilon.-(13-carboxytridecanoylammo)Lys.sup.20]exendin-4- -NH.sub.2; [N.sup..epsilon.-(11-carboxy-undecanoylamino)Lys.sup.20]exendin- -4-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-MPA)-NH.sub.2; exendin-4-Lys.sup.40(s-AEEA-AEEA-MPA)-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-AEEA-MPA)-NH.sub.2; exendin-4-Lys.sup.40(.epsilon.-MPA)-albumin; exendin-4-Lys.sup.40(.epsilon.-AEEA-AEEA-MPA)-albumin; exendin-4-Lys.sup.40(.epsilon.-AEEA-MPA)-albumin; GLP-1(7-38); desamino-His.sup.7,Arg.sup.26, Lys.sup.34(.gamma.-Glu(N-.alpha.-hexadecanoyl)))-GLP-1(7-37); desamino-His.sup.7,Arg.sup.26,Lys.sup.34,(N.sup..epsilon.octanoyl)-GLP-1(- 7-37); Arg.sup.26,34,Lys.sup.38(N.sup..epsilon.-(.omega.carboxypentadecano- yl))-GLP-1(7-38); Arg.sup.26,34,Lys.sup.36(N.sup..epsilon.-(.gamma.-Glu(N-.alpha.-hexadecan- oyl)))-GLP-1(7-37); or any peptide comprising the amino acid sequence of any one of SEQ ID NOs:1-40 and 43-45; wherein the peptide comprising the amino acid sequence of any one of SEQ ID NOs:25-40 may optionally be linked to the amino acid sequence of SEQ ID NO:42; wherein MPA and AEEA refer to maleimidopropionic acid and [2-(2-amino)ethoxy)lethoxy acetic acid, respectively; and wherein the test compound is optionally amidated. 17. A method for detecting or measuring GLP-1 receptor agonist activity of a GLP-1 receptor agonist compound comprising the steps of: (a) preparing a sample comprising a GLP-1 receptor agonist compound; 6-23 (clone 6) cells; and cAMP linked to a fluorescent moiety capable of emitting fluorescence at a correcting wavelength of about 665 nm; (b) adding an agent to stimulate the sample from step (a) to produce cAMP in the 6-23 (clone 6) cells; (c) forming a reaction mixture by combining (i) the sample from step (b); and (ii) an anti-cAMP monoclonal antibody linked to Europium-cryptate capable of generating emitted fluorescence at a measuring wavelength of about 620 nm; wherein the reaction mixture comprises a buffer capable of lysing the 6-23 (clone 6) cells; (d) irradiating the reaction mixture from step (c) at a single excitation wavelength of about 337 nm by an external radiation source; (e) simultaneously measuring both the emitted fluorescence at about 620 nm and the emitted fluorescence at about 665 nm which takes account interference parameters of the reaction mixture; (f) calculating a corrected fluorescence for the fluorescence emitted by the compound at about 620 nm based on the fluorescence emitted by the compound at about 665 tun; (g) correlating the corrected fluorescence reading to the presence or quantitative amount of cAMP in the sample; and (h) correlating the amount of cAMP in the sample to the GLP-1 receptor agonist activity of the GLP-1 receptor agonist compound. 18. The method of claim 17, wherein the sample comprises: (i) a pharmaceutical composition which comprises the GLP-1 receptor agonist compound, wherein the GLP-1 receptor agonist compound is exenatide; (ii) 6-23 (clone 6) cells; and (iii) cAMP linked to a fluorescent moiety capable of emitting fluorescence at a correcting wavelength of about 665 nm. 19. The method of claim 17, further comprising incubating the reaction mixture. 20. The method of claim 17, wherein the GLP-1 receptor agonist compound is exenatide.

Details for Patent 8,603,761

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Glaxosmithkline Llc	TANZEUM	albiglutide	For Injection	125431	04/15/2014		2028-05-23
Sanofi-aventis U.s. Llc	ADLYXIN	lixisenatide	Injection	208471	07/27/2016		2028-05-23